ABSTRACT The relationship between growth factors and breast cancer etiology is complex and not completely understood; however, strong experimental evidence supports their role in breast carcinogenesis. Therefore, in this proposal, we will conduct a detailed evaluation of prolactin and its association with breast cancer risk. This study will use a prospective nested case-control design among pre- or perimenopausal women at blood collection (1,542 cases diagnosed through 2011). Samples were collected in the Nurses' Health Study II (NHSII) in 1996-99 from 29,611 women, ages 32 to 52 years, and in 1989-90 from 32,826 NHS participants, ages 43 to 62 years. In addition to assessing the overall prolactin-breast cancer association, this study will examine whether prolactin is more strongly associated with risk of estrogen receptor positive tumors, as well as tumors staining positive for prolactin, prolactin receptor, cyclin D1, and phosphorylated STAT3 and STAT5. Observing such specificity to the prolactin receptor and its downstream targets would add substantially to the case for causality. Prior studies have assessed prolactin levels using an immunoassay that measures multiple isoforms with differing biologic activities. This study will provide the first prospective assessment of breast cancer risk with a prolactin bioactivity assay, which may better reflect the portion of prolactin that is biologically important. Also, since few correlates of bioactive prolactin are known, this proposal will assess, for the first time, its relationship with known and putative breast cancer risk factors (e.g., parity, shift work). The availability of existing hormone data for this cohort will enable the examination of prolactin as an independent breast cancer risk factor and its interrelationships with other hormones. Importantly, we have added an aim to directly evaluate the addition of prolactin to a comprehensive risk prediction model for breast cancer, which could be useful clinically. Several features of prolactin make it an attractive candidate for inclusion into such models, including that the immunoassay is a standardized and common clinical assay that is very inexpensive and easy to measure. Further, the magnitude of the prolactin association appears to be similar to other factors included in breast cancer risk prediction models. We also will evaluate the inclusion of the bioassay; if this improves prediction better than the immunoassay, it would provide impetus to identify the specific isoforms of prolactin that increase risk of breast cancer and develop inexpensive assays for their measurement. Other grants will provide funding for follow-up and other hormone assays, increasing the cost-effectiveness of this project. Ultimately, elucidating the role of prolactin in breast cancer could open up multiple new areas of research for both treatment and prevention.